Figure 6.

CSN5i-3 increases vascular leakage in vivo. (A) Zebrafish Tg(Fli1:GFP)y1 casper embryos were treated with 1, 10, 20 and 50 μM CSN5i-3, 10 μM MLN4924 or solvent (DMSO 0.5%) for 48 hours from 24 hpf onward. Embryos were lysed and western blot analysis of Cullin-3 was performed. ERK 1/2 was used as loading control. Blot images were cropped for clarity of presentation (full blots are in Supplemental Fig. 12). (B) Representative images of vascular leakage in control vs CSN5i-3 treated zebrafish embyros. Zebrafish embryos were treated with 50 μM CSN5i-3 or solvent (DMSO 0.5%) from 1 dpf onward for 72 hours. 70 kDa TMR-dextran was injected at the intersection of the common cardinal vein, the posterior cardinal vein and the primary head sinus to visualize vascular leakage. 70 kDa Dextran is in red, vasculature in green. Scale bar represents 50 μm. (C) Quantification of relative extravascular fluorescence from 0.5% DMSO (n = 11) or 50 μM CSN5i-3 (n = 9) treated fish. For each embryo, the fluorescence intensity of the dextran was measured in four intersegmental vessels and three intervascular areas between the intersegmental vessels. The average of the dextran fluorescence in the intervascular areas was normalized to the average dextran fluorescence inside the vessels. Obtained values represent the ratio of extravasated dextran compared to dextran inside the vessels. ***p < 0.001 compared to control in student’s t-test.